The position of a gene along a chromosome can influence its expression. Such position effects play key roles in the regulation of gene expression and determination of cell fate in a wide variety of organisms from human to yeast. Position effects usually inactivate gene expression over large distances by a mechanism that is thought to involve changes in chromatin packaging. The budding yeast, S. cerevisiae, offers a genetically well defined system for studying this phenomenon. The yeast SIR2, SIR3, and SIR4 proteins are known from genetic studies to be involved in generation of inactive chromatin domains, but their mechanism of action is unknown. The goal of this proposal is to use the SIR proteins as a starting point towards the biochemical dissection of the structure of inactive chromatin domains. Thus, the SIR proteins will be purified, their interacting factors identified, and these purified components will be used to reconstruct inactive chromosome domains in vitro. This system will then provide the basis for biochemical experiments to deduce molecular mechanisms. Given the high degree of conservation of gene regulatory proteins and the general similarity of position effects in yeast and other eukaryotes, it seems likely that many of the principles developed for the yeast proteins covered in this proposal will apply in other settings. A basic understanding of the molecular events that control cell fate provides not only a frame work for understanding how the process can fail, but also provides the substrates and knowledge to design therapeutic strategies based on intervention.